Edible products with reduced oxidation and spoilage

ABSTRACT

The invention is based, in part, on the discovery that the addition of cations, such as calcium or magnesium ions, to muscle tissue before solubilization of the muscle proteins enhances removal of membranes, which reduces oxidation and spoilage of the muscle tissue.

TECHNICAL FIELD

This invention relates to a process for isolating edible protein fromanimal muscle by removing membranes to reduce oxidation and spoilage ofthe edible protein.

BACKGROUND

Low value animal muscle (e.g., from fatty pelagic fish or poultry boneresidue) is usually undesirable as a source of food for humanconsumption. After processing, the isolated protein is oftencharacterized by unattractive textures, dark colors, and strong flavors.Such unfavorable characteristics are often due to membrane lipidoxidation. The problem is particularly acute with fish since theirlipids are susceptible to oxidation partly because of the highlyunsaturated nature of the fatty acids.

One impediment to implementing many methods of food processing, e.g.,protein extraction, on an industrial scale has been the cost ofcentrifuging solubilized proteins to remove the membranes in industrialequipment. Standard industrial decanter centrifuges typically apply agravitational force of up to approximately 4,000 g, which is generallyineffective.

SUMMARY

The invention is based, in part, on the discovery that the addition ofspecific cations, such as calcium or magnesium ions, and optionally anorganic acid, to muscle tissue before solubilization of the muscleproteins can enhance removal of membranes, which significantly reducesoxidation and spoilage of the muscle tissue.

In general, the invention features a method for isolating edible proteincompositions from animal muscle by (a) obtaining a mixture comprisingminced or ground animal muscle, (b) adding an amount of a polyvalent,food-grade cation to the mixture sufficient to separate cellularmembranes from cytoskeletal proteins in the animal muscle, and (c)treating the mixture to reduce the oxidation potential of the separatedcellular membranes. The method can optionally include a step ofhomogenizing the animal muscle.

In this method, the polyvalent, food-grade cations are metallic cations,e.g., calcium and magnesium ions, and the final concentration of calciumand/or magnesium ions in the mixture can be in a range of about 0.1 mMto about 50 mM after the addition of an aqueous solvent such as water.Magnesium and calcium ions can be added to the mixture by, e.g., addingmagnesium chloride or calcium chloride, respectively, to the mixture.Following the addition of ions, the mixture can optionally be incubatedfor a period of time, e.g., from 3 up to 60 minutes, or more, e.g., 5,10, 15, or 30 minutes.

Treating the mixture can include aggregating at least a portion of theseparated cellular membranes in the mixture to reduce the totalseparated membrane surface area, thereby reducing the oxidationpotential. Cellular membranes can be aggregated, e.g., by incubating themixture after the addition of cations. As another example, cellmembranes can be aggregated by adding an acid to the mixture. As stillanother example, cell membranes can be aggregated by adding an aggregantto the mixture. Suitable aggregants include carrageenan, algin,demethylated pectin, gum arabic, chitosan, polyethyleneimine, spermine,spermidine, calcium salt, magnesium salt, sulfate, phosphate, andpolyamine. In certain embodiments of the present invention, steps (b)and (c) are performed simultaneously by adding a sufficient amount ofcalcium or magnesium ions to cause both separation of cellular membranesand cytoskeletal proteins and aggregation of the membranes.

The method can also include adjusting the pH of the mixture tosolubilize at least a portion of the protein in the mixture.Solubilization of the proteins can be performed by lowering the pH ofthe mixture, e.g., by adding an acid to the mixture. Where an acid isused to adjust the pH of the mixture, an amount sufficient to lower thepH to below about 3.5 can be added. Solubilization can also be performedby increasing the pH of the mixture, e.g., by adding a base to themixture. Where a base is used to adjust the pH of the mixture, an amountsufficient to raise the pH to greater than about 10.0 can be added.

If the proteins are solubilized, the method can further includeseparating cell membranes from the solubilized protein. Separation canbe performed by centrifugation, e.g., by centrifuging the mixture atfrom about 500×g to about 10,000×g. Alternatively or in addition,separation can be performed by precipitation. The solubilized proteincan be collected from the mixture. An aggregant can be added toaggregate membranes and facilitate their removal followingsolubilization of the proteins. The aggregant can be, e.g., carrageenan,algin, demethylated pectin, gum arabic, chitosan, polyethyleneimine,spermine, spermidine, calcium salt, magnesium salt, sulfate, phosphate,and/or polyamine.

The method can include dewatering the mixture. Dewatering can performed,e.g., by centrifuging, filtering, and/or pressing the mixture (e.g.,using a French press).

In another aspect, the present invention includes a protein compositionproduced using any of the methods described above.

In another aspect, the invention includes an edible protein compositionwith reduced oxidation potential that includes minced or ground animalmuscle, wherein the composition comprises a cellular membrane content ofless than 40% (e.g., less than 30, 25, 20, 10, 5, or 1%) of a cellularmembrane content in a sample of the animal muscle as removed from theanimal prior to mincing or grinding, or other processing.

In still another aspect, the invention includes an edible proteincomposition with reduced oxidation potential, which includes minced orground animal muscle, wherein the composition comprises a cellularmembrane content approximately equal to the cellular membrane content ina sample of the animal muscle as removed from the animal prior tomincing or grinding, but wherein at least 60% (e.g., 70, 75, 80, 90, 95,or 99%, or all) of the cellular membranes in the edible proteincomposition are separated from cytoskeletal proteins and are inaggregated form, as determined using microscopy, e.g., in conjuctionwith immunological techniques.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The present invention relates to a simple, inexpensive, and effectiveway of treating animal muscle to significantly improve the efficiency ofmembrane removal from solubilized proteins. The resulting edible proteincomposition has a significantly reduced oxidation potential, i.e., isrelatively free of cellular membranes that can become oxidized and causespoilage of the composition, is capable of forming a gel, and can beprocessed into human and animal foods.

Sources of Protein

The process of the present invention can be used to prepare proteinisolates from any protein source. For example, any flesh that isrecovered from fish, e.g., fillets, or any portion of the fish leftafter the fillets have been removed (e.g., portions that are nottypically used for human food), can be processed. Similarly, there isvery little usage of the skeletons of chickens after parts are removedfor retail sale. The methods of the present invention can process suchchicken and fish parts to produce edible protein suitable for humanconsumption. Other underutilized muscle sources useful in the methods ofthe invention include Antarctic krill, which is available in largequantities but is difficult to convert to human food because of itssmall size.

Representative suitable starting sources of animal muscle for theprocesses of this invention include fish fillets, deheaded and guttedfish, crustaceans (e.g., krill), mollusks (e.g., squid), chicken andother poultry (e.g., turkey), beef, pork, or lamb. The present inventionfurther contemplates that plant material, e.g., soy, can also be asuitable starting source of protein, and that the methods describedherein can be use to produce plant protein isolates.

Treatment of Animal Muscle Prior to Alkaline or Acid Extraction

Animal muscle is processed to separate cellular membranes fromcytoskeletal proteins and is then treated to reduce the oxidationpotential of the cellular membranes, and thus of the muscle mixture orresulting protein composition. The separated cellular membranes can betreated by incubating the mixture for a sufficient time to allow themembranes to aggregate, thereby reducing the total surface area of themembranes. The addition of acids and/or aggregants enhances thisprocess. Alternatively, the membranes can be removed.

Animal muscle is minced or ground, and is optionally mixed with anaqueous solution. The animal muscle can represent any percentage byweight of the mixture, and can be a relatively low percentage, e.g.,less than about 15% (e.g., 5, 6, 8, 10, or 12%) by weight of themixture. Any aqueous solvent, e.g., water, can be used. In addition, themuscle can be washed with an aqueous solution prior to any mechanicalmanipulation. The muscle can be substantially diluted in water such thatthe solubilized protein suspension/solution produced in successive stepsof the method is of a low enough viscosity to enhance the removal oflipids or insoluble material, e.g., by centrifugation. Lower viscositycan also aid removal of mixture components using methods other thancentrifugation, as described herein. The viscosity of the proteinsuspension/solution is preferably about 75 mPa·s or less (e.g., about65, 60, 50, 45, 40, 35, or 30 mPa·s or less). Viscosity is measured, forexample, with a Brookfield Model LVF viscometer (Brookfield Engineering,Stoughton, Mass.) as directed by the manufacturer. The manufacturer'ssupplied conversion chart is then used to calculate viscosity. Theanimal muscle can be mechanically ground, or minced, or chopped by hand.Thereafter, the minced or ground muscle can be further homogenized tocreate very fine particles.

After dilution of the animal muscle with water or an aqueous solution toform a mixture, the pH level of the mixture should be about 4 to about10, e.g., about 6.0 to about 8.0, or about 6.5 to about 7.5, or about6.8. The pH can be adjusted to get the mixture within this range.

In the next step, polyvalent food-grade cations, e.g., from metal salts,such as calcium ions (e.g., in the form of calcium chloride) ormagnesium ions (e.g., in the form of magnesium chloride) are added tothe mixture in an amount sufficient to separate cellular membranes fromthe cytoskeletal proteins in the animal muscle. A single type of ion ormixtures of different types of ions can be added. For example, a mixtureof calcium and magnesium ions can be added.

Alternatively or in addition, dry metal salts, e.g., calcium chloride ormagnesium chloride, can be added to dry starting material, such asminced animal muscle, or animal muscle in any form that has not yet beenmixed with water or an aqueous solvent. Where this is the case, thewater or aqueous solvent is added in a step subsequent to the additionof ions, e.g., to prepare a mixture for solubilization of the proteins.

As used herein, the term “food-grade cations” means cations suitable forhuman consumption. Guidelines for determining whether a source of ions,e.g., calcium chloride or magnesium chloride, is food-grade are providedby the United States Food and Drug Administration (FDA). Adding ions tothe mixture as described above causes cellular membranes to separatefrom their respective cytoskeletal proteins, facilitating removal and/oraggregation of the membranes. Aggregation of the membranes lowers theiroxidation potential, e.g., by reducing the oxidizable surface area ofthe membranes. Removing the separated membranes also reduces theiroxidation potential.

When using calcium or magnesium ions in the new methods, the finalconcentration of calcium and/or magnesium ions in the mixture should bein the range of about 0.1 mM to about 50 mM, e.g., about 1 mM to about45 mM, about 10 mM to about 40 mM, about 20 mM to about 35 mM, or about25 mM to about 30 mM. It is also contemplated that concentrations ofgreater than 50 mM can be used. When ions are added to dry startingmaterial, the final concentration of ions in the mixture is measuredfollowing creation of the mixture, i.e., following addition of water oraqueous solvent to the dry starting material. Skilled practitioners willappreciate that the amount of ions present in a mixture is bestexpressed in terms of molarity because this measurement expresses thefinal concentration of reactive ions in the mixture, i.e., the amount ofions available to cause separation of cellular membranes fromcytoskeletal proteins.

Calcium or magnesium chloride can be obtained from any source, e.g., acommercial source that supplies such compounds for use in foods, or formedical, experimental, or industrial uses.

Optionally, the mixture can be incubated for a period of time followingthe addition of ions to the mixture. The mixture can be incubated forany period, e.g., less than about 1 minute to about 180 minutes, e.g.,about 5 to about 150 minutes, about 10 to about 120 minutes, about 20 toabout 90 minutes, or about 30 to about 60 minutes. In variousembodiments of the present invention, the incubation period is about 0,3, 5, 10, 30, or 60 minutes long. The present invention contemplatesthat even periods of time longer that 180 minutes can be utilized.

Optionally, an aggregant can be added to the mixture to cause/facilitateaggregation of the cellular membranes. Suitable polymer aggregantsinclude carrageenan, algin, demethylated pectin, gum arabic, chitosan,polyethyleneimine, spermine, and spermidine. For example, chitosan canbe added to the protein in an amount ranging from 0.1 to 1.0% by weightof the protein. Other aggregants include salts, such as a calcium andmagnesium (which can be added in amounts additional to those describedabove for causing separation of cellular membranes and cytoskeletalproteins), sulfate and phosphate (which can be added in amounts equal tothose described above for magnesium and calcium for causing separationof cellular membranes and cytoskeletal proteins), and polyamine. Skilledpractitioners will recognize that a sufficient amount of calcium and/ormagnesium salts can be added during a the first addition of thesecalcium and/or magnesium ions (described above) to cause both cellularmembrane/cytoskeletal protein separation and aggregation of themembranes.

Optionally, an organic acid, such as citric acid, or citrate salt, isadded to the mixture before, during, and/or after the addition of ionsto the mixture. Further, citric acid can be added before, during, and/orafter any period of incubation of the mixture with calcium or magnesiumchloride, and can be added to the mixture such that the finalconcentration of citric acid in the mixture is from about 0.001 mM toabout 10 mM, e.g., from about 0.01 mM to about 9 mM, about 0.1 to about5 mM, about 0.5 to about 2 mM, e.g., about 1 mM. Alternatively or inaddition, other organic acids can be used in the methods of the presentinvention, e.g., malic, maleic, and/or tartaric acid, fumaric, or saltsthereof. Addition of an organic acid to the mixture during the processimproves the effect of adding ions to the mixture, causing greater andmore rapid separation of cellular membranes from cytoskeletal proteins.Further, the present invention contemplates that inorganic acids, e.g.,sulfuric or any other polyanionic acid, can be used in addition, oralternative to, the use of an organic acid.

When ions and an organic acid, e.g., citric acid, are used incombination as described above, the process can be terminated followingthe addition of ions and the acid to the mixture, because the cellmembranes will have separated and aggregated. The mixture can then bedewatered using any method known in the art, e.g., by pressing, toremove/reduce the water content of the mixture. Pressing can beperformed, for example, using a French press or by centrifuging themixture. As another example, the mixture can be filtered to removewater.

Alternatively, whether or not an organic acid is added to the mixture,the pH of the mixture can then be adjusted to an alkaline pH or anacidic pH for solubilization of the proteins and/or removal of themembranes from the mixture, as described in further detail below.

Acid and Alkaline Extraction of Proteins

To solubilize a portion, e.g., at least 50%, e.g., at least 60, 70, 75,80, 85, or 90%, of the animal protein by weight, the pH of the mixtureis increased or decreased. For example, the pH of the mixture can beincreased, e.g., to greater than about 10.0 (e.g., about 10.0 to 11.0 or11.5, or about 10.5). Alternatively, the pH of the mixture can bedecreased, e.g., to below about 3.5 (e.g., about 3).

Protein denaturation and protein hydrolysis is a function of temperatureand time in solution, with increasing temperature and time in solutionpromoting protein denaturation and hydrolysis. The aqueous compositionalso may contain components such as preservatives, which protectproteins from degradation. In addition, the ionic strength of thesolution can be adjusted to avoid protein precipitation.

In the acid and alkaline extraction methods, any acid or base,respectively, that does not contaminate the final product can be used tolower the pH of a mixture. For example, organic acids (e.g., malic acidor tartaric acid) or mineral acids (e.g., hydrochloric acid or sulfuricacid) are suitable. Citric acid which has a favorable pK_(a) value canprovide buffering capacity at pH 3 and pH 5.5. Acids that havesignificant volatility and impart undesirable odors, such as acetic acidor butyric acid, are undesirable. Likewise, any of several bases, e.g.,NaOH, can be used to raise the pH of the mixture. Polyphosphates aresuitable, since they also function as antioxidants and improve thefunctional properties of the muscle proteins.

Since the control of the pH of a mixture can often be difficult, themixture can include a buffer that maintains an acidic target pH value ora basic target pH value. Given a target pH, the choice of buffer iswithin the skill in the art of food science. Buffers suitable for atarget pH in the range of 8.0 to 9.0 include glycine, arginine,asparagine, cysteine, carnosine, taurine, pyrophosphate, andorthophosphate. Buffers suitable for a target pH in the range of 5.5 to6.5 include histidine, succinate, citrate, pyrophosphate, and malonate.Buffers suitable for a target pH in the range of 2.0 to 2.5 includealanine, glutamic acid, citric acid, lactic acid, phosphoric acid, orpyruvic acid.

Removal of Cellular Membranes

Optionally, cellular membranes can be removed physically from themixture to reduce their oxidation potential (and thus the oxidationpotential of the entire mixture). Cellular membranes are removed, e.g.,by centrifugation, after the proteins in the mixture have beensolubilized as described above.

For example, the mixture can be centrifuged so that the charged membranelipids are separated from an aqueous phase, which is collected by, forexample, decanting the aqueous phase. Using the new methods, the mixturecan be centrifuged, e.g., at from less than 500×g to about 10,000×g, orhigher, e.g., at 1000×g, 2000×g, 3000×g, 4000×g, 6000×g, 7000×g,8,000×g, or 9,000×g. Several layers can form after centrifugation. Atthe bottom, the charged membrane lipids and any remaining residue arepelleted. The percentage sediment weight can be less than 20% (e.g.,less than 10%), and higher sediment percentage may indicate that some ofthe desirable protein has been removed with the undesirable lipids.Percentage sediment weight is defined as the weight of pellet aftercentrifugation divided by the total homogenate mixture weight. Above thepellet is an aqueous layer containing the solubilized protein. At thetop, the neutral lipids (fats and oils), if any, float above the aqueouslayer. The neutral lipids can be removed with a pipette before decantingthe aqueous phase. Intervening layers can also be present depending onthe source of muscle. For example, a gel of entrapped water containingsolubilized protein can form between the aqueous layer and the pellet.This gel can be kept with the aqueous layer to increase protein yield.

Of course, in industrial applications, the aqueous phase (and otherphases, if desired) can be removed during centrifugation using acontinuous-flow centrifuge or other industrial scale machinery.

Methods other than centrifugation can be used to separate the membranelipids from the aqueous phase. For example, a variety of filtrationdevices are available to the skilled artisan, depending on the size andvolume of the material to be separated. If the membranes are notaggregated, a microfiltration apparatus is suitable and can be used forseparating molecules having molecular weights in the range of 500,000 to20 million. If the membranes are aggregated, particulate filtration maybe suitable. These filtration units typically operate under pressure inthe range of 2 to 350 kPa. In addition, cationic exchange membranes(sc-1) and anionic exchange membranes (sa-1) are suitable for removingmembrane lipids from the mixture. In addition, various filtrationmethods can be used to select for or against muscle proteins of aparticular size.

In some circumstances, an HF-lab-5 ultrafiltration unit (Romicon, Inc.,Woburn, Mass.) can be used with a feed tank having an immersed coolingcoil to maintain a relatively constant temperature. A cross flowprocess, which has the advantage of removing filter cake continuously,can also be used. To recover water or lower the salt content of themixture, filtration membranes can be used with electrodialysis to driveout ions from the mixture. For this particular purpose, a stackpack unit(Stantech, Inc., Hamburg, Germany) can be used. This unit containsseveral cell pairs sandwiched between two electrode compartments.

Removal of membranes can also be facilitated by subjecting a mixture tohigh pressure, using, e.g., the MPF 7000 device (Mitsubishi HeavyIndustries, Ltd.) or the High Pressure ACB 665 device (Gec, Alsthom;Nantes, Frances). High pressure treatment, accompanied by the propertemperature treatment, has the added benefit of killing known pathogens,in addition to aggregating and separating membrane lipids.

To further facilitate removal of membranes after solubilization of theproteins, an aggregant can be added to the mixture. As discussed above,suitable compounds for aggregating membranes include polymer aggregants,such as carrageenan, algin, demethylated pectin, gum arabic, chitosan,polyethyleneimine, spermine, and spermidine. Other aggregants includesalts, such as a calcium salt, magnesium salt, sulfate, phosphate, andpolyamine.

Alternatively or in addition, the protein in the protein-richsupernatant can be recovered (i.e., removed from the cellular membranes)by adjusting the pH to a level at which at least some of the proteinsprecipitate. This pH will vary depending upon the source of the proteinand can be between about 5.0 and about 7.5, e.g., between about 5.3 toabout 7.3, about 5.5 to about 7.0, or about 6.0 to about 6.8, e.g., 6.8.For example, the pH can be between about 5.3 and about 5.5. The yieldcan be at least 70% (e.g., at least 90%) by weight of the total startingprotein in the mixture. The yield is defined as the precipitated proteinmass divided by the total protein mass (e.g., as determined by theMarkwell method) of the starting material. Concentration of protein canbe measured by any method known in the art, e.g., by the Markwell method(a modified Lowry assay). Cryoprotectants (e.g., disaccharides and/orpolyalcohols, such as polysorbates) can be added to the precipitatedprotein to preserve and protect the product during freezing and storage.

Alternative or in addition to adjusting the pH of the solution to causeprotein precipitation, polymers such as polysaccharides, chargedpolymers, marine hydrocolloids including alginates or carrageenan or thelike, can be added, either alone or in combination with centrifugation.Such compounds can be added before, after, or instead of, adjusting thepH of the mixture to precipitate proteins. The salt concentration of theaqueous phase can also be adjusted to facilitate precipitation.

Skilled practitioners will appreciate the washes, supernatants, andflow-through fractions described above can be recycled back to earliersteps to recover even more protein using the methods. For example, aftersolubilized protein has been precipitated, the aqueous fraction can beentered into another batch of animal muscle that has yet to besolubilized.

Edible Protein Compositions

The methods described herein can be used to produce edible proteincompositions having advantageous properties. For example, the proteincompositions have a relatively low cellular membrane content. Dependingupon the source (e.g., fish, beef, or chicken) of the starting material,the methods described herein can remove about 70% to 90% of cellularmembranes from a sample of starting material at a relatively lowcentrifugation speed (e.g., 4000×g). Higher percentages can be removedusing higher centrifugation speeds. Accordingly, a practitioner usingthe new methods can obtain protein compositions containing less than30%, e.g., less than 25%, 20%, 15%, 10%, 5%, or 1%, or beingsubstantially free of cellular membranes, as compared to the cellularmembrane content of an equal amount of starting material.

Further, the protein compositions described above can have a cellularmembrane content that is relatively resistant to oxidation. In themethods of the present invention, ions (e.g., magnesium and/or calcium)are added to a mixture containing the starting material to separatecellular membranes from cytoskeletal proteins in the material.Thereafter, lowering the pH of the mixture (e.g., to below pH 6.5) byadding acid, or adding an aggregant, to the mixture at any step of theprocess causes the cellular membranes to aggregate, reducing theirsurface area and rendering them less susceptible to oxidation. Dataillustrating the reduced susceptibility of aggregated cell membranes tooxidation is provided below in Example 11. The extent to which thecellular membranes aggregate is pH- and time-dependent, i.e., a lower pHcauses more rapid aggregation of membranes than does a higher pH.

Skilled practitioners will appreciate that aggregated cellular membranesare less likely to adversely affect the quality of the proteincomposition containing them, and that such protein compositions have anextended shelf-life. The shelf-life of such protein compositions can beextended further by any art-known method, e.g., by cooking, freezing,and/or adding antioxidants to the composition.

If aggregated, cellular membranes need not be removed from the proteincomposition, because the total membrane surface area is sufficientlyreduced to greatly lower the rate of oxidation. Accordingly, aggregatedcellular membranes can remain in the protein composition because theyare less susceptible to oxidation than non-aggregated membranes. Thus,the present invention includes protein compositions having a cellularmembrane content similar to that of the starting material, wherein atleast a portion of the membrane content is in aggregated form and,therefore, less susceptible to oxidation than an equivalent amount ofnon-aggregated cellular membrane. Such protein compositions can beexpected to have a 2-fold, 3-fold, 4-fold, or more, extended shelf life,as compared to raw muscle meat not processed according to the methods ofthe present invention. Skilled practitioners will be able to determinewhether the cellular membrane content of a protein composition iswholly, or in part, in aggregated form using any art-known method, e.g.,microscopy, especially in conjunction with immunological techniques.

The cellular membrane content in a protein composition prepared usingthe methods of the present invention can be determined using any methodknown to those of skill in the art. For example, total phospholipidcontent in the protein composition can be measured and compared to thetotal phospholipid content of an equal amount of untreated startingmaterial. Similar methods were used in the working examples describedbelow. As another example, specific membrane-associated proteins, e.g.,calcium ATPase, can be used as “markers” to determine the amount ofcellular membrane present in a protein composition. Total marker contentcan be measured in both the protein composition and the untreatedstarting material, and the results can be compared to determine theamount of cellular membrane remaining in the protein composition.Skilled practitioners will appreciate that there is a direct linearcorrelation between cellular membrane content and content of anyparticular membrane-associated protein. If membranes are aggregatedrather than removed, this condition can be observed using a lightmicroscope.

Use of Edible Protein

The new methods can be used to process protein from any source. Forexample, the methods can be used to process for human consumptionmaterials that are not presently used as human foods due to theirinstability and unfavorable sensory qualities. Small pelagic species offish such as herring, mackerel, menhaden, capelin, anchovies, orsardines are either underutilized or used for nonhuman uses.Approximately one half the fish presently caught in the world are notused for human food. A process that produces an acceptable stableprotein concentrate opens the use of such material for humanconsumption.

The invention will be further described in the following examples, whichdo not limit the scope of the invention defined by the claims.

EXAMPLES Example 1 The Effect of pH on Sedimentation of SeparatedMembrane

The effect of pH on the sedimentation behavior of isolated musclemembranes was determined to see how differently isolated membranesbehaved from those that were in the presence of solubilized protein ateither high or low pH.

The isolated membranes were first prepared as follows:

1. Fish fillet was ground.

2. Four volumes of histidine buffer (0.12M KCl, 5 mM histidine, pH=7.3)were added to the ground muscle.

3. The mixture was homogenized by a Polytron® homogenizer with twobursts of 30 seconds.

4. The homogenate was centrifuged at 6,000 g for 20 minutes, and thesupernatant was kept.

5. The supernatant was then centrifuged again at 50,000 g for 20minutes. The sediment was kept after centrifugation.

6. The sediment was resuspended in 0.6 M KCl and centrifuged at 50,000 gfor 20 minutes to remove actomyosin.

7. The resultant sediment was resuspended in the histidine buffer, whichwas defined as “membrane fraction.”

8. The concentration of proteins in the membrane fraction was measuredby the Markwell method (an art-recognized, modified Lowry assay).

9. Recovery of membrane in the experiments below was determined byprotein in the isolated membrane fractions and by phospholipid (PL) inthe solubilized muscle preparations.

The separated membrane suspensions were adjusted to pH 3-10.5respectively. The pH-adjusted membrane suspensions were then centrifugedat 4,000×g for 15 minutes. The remaining protein content in theresultant supernatants was measured to estimate the percent of membraneremaining in the supernatant after centrifugation. The results aretabulated in Table 1, below, and show that separated membrane can besedimented efficiently with low pH treatment, while almost all “high pHtreated membrane” remains suspended in the buffer after centrifugation.TABLE 1 The pH Effect on Membrane Sedimentation (percent membraneremaining in the supernatant after centrifugation). pH 3 4 5 6 7 8 9 1010.5 % 2.45 2.13 3.62 76.39 93.99 82.66 89.22 95.78 98.23

Studies of the following pH treatments were performed (centrifugationparameter is 4,000 g for 15 minutes in all cases) to investigate the lowpH effect on sedimentation of separated membrane.

Treatment:

-   -   a. Start at pH 7.3, adjust to low pH (3-6), subject to        centrifugation.    -   b. Start at pH 7.3, adjust to 10.5, adjust to low pH (3-6),        subject to centrifugation.    -   c. Start at pH 7.3, adjust to low pH (3-6), adjust to 10.5,        subject to centrifugation.

d. Start at pH 7.3, adjust to 10.5, adjust to low pH (3-6), adjust to10.5 centrifugation. TABLE 2 The Percent (%) of Protein Remaining in theSupernatant after Centrifugation low pH used pH 3 pH 4 pH 5 pH 6 a 2.45%2.13% 3.62% 76.39% b 2.34% 1.53% 3.09% 83.27% c 8.71% 9.79% 13.28%91.95% d 8.25% 32.58% 82.90% 94.09%

TABLE 3 The Percent of Lipid Phosphorous Remaining in the Supernatantafter Centrifugation Low pH used pH 3 pH 4 pH 5 pH 6 a <2.00% <2.00%<2.00% 68.62% b <2.00% <2.00% <2.00% 75.18% c <2.00% <2.00% <2.00%82.26% d <2.00% 31.62% 73.76% 94.61%

The data above indicate that values of pH below pH 6 were effective insedimenting the suspended membranes at 4,000×g and 15 minutes. Removaldid not take place at high pH, e.g., 10.5. However, effective removal atpH 10.5 could be accomplished if the muscle protein was first treated atlow pH and then brought to pH 10.5.

Example 2 Sedimentation of Separated Membranes Using Centrifugation

Separated membrane suspensions were first adjusted to pH 3 or 4. The pHadjusted membrane suspensions were then centrifuged at different gforces (from 500×g to 2,500×g) for 15 minutes. The remaining membraneprotein content in the supernatants after different g forcecentrifugation was measured. The results are provided in Table 4, below.TABLE 4 The Effect of g Force on Membrane Sedimentation 2500 g 2000 g1500 g 1000 g 500 g pH = 3 2.51% 2.88% 5.33% 7.01% 29.77% pH = 4 2.47%2.09% 2.41% 3.62% 22.14%

The data in Table 4 indicate that the low pH-adjusted membranes wereeasily sedimented from suspension by g forces as low as 1,000×g and evenat 500×g.

Example 3 Proteins Remaining in Supernatant after Centrifugation

The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)profiles of separated membrane and membrane proteins remaining in thesupernatant after low pH treatments and centrifugation wereinvestigated. The results are tabulated in Table 5. Since the membranestudied was prepared from 100 g cod muscle tissue and the membraneprotein was 108 mg, the data in the Table 5 is expressed as milligramsof protein remaining in the supernatant from the original total amountof 108 mg membrane protein prepared from 100 g cod muscle. Samples wereprepared as follows:

-   Sample A: Membrane suspension-   Sample B: Supernatant of membrane suspension after pH 7.3 to 3 to    10.5 treatment-   Sample C: Supernatant of membrane suspension after pH 7.3 to 10.5 to    3 to 10.5 treatment-   Sample D: Supernatant of membrane suspension after pH 7.3 to 5    treatment-   Sample E: Supernatant of membrane suspension after pH 7.3 to 5 to    10.5 treatment-   Sample F: Supernatant of membrane suspension after pH 7.3 to 10.5 to    5 treatment-   Sample G: Supernatant of membrane suspension after pH 7.3 to 10.5 to    5 to 10.5 treatment

The protein remaining in the supernatant after pH 7.3 to 3 and 7.3 to10.5 to 3 treatments was too little to be detected by SDS-PAGE. TABLE 5The Profiles of Proteins Remaining in the Supernatant after DifferentLow pH Treatment. Mw Sample A Sample B Sample C Sample D Sample E SampleF Sample G (kDa) (mg) (mg) (mg) (mg) (mg) (mg) (mg) 260 3.24 2.82-3.290.24-0.40 0.82 0.43 0.83-0.90 8.95 230 7.56-8.64 0.94-1.22 0.48-0.560.98-1.25 0.57-0.71 0.43-0.80 10.74  200 7.56-10.8 0.66-1.41 3.210.51-0.55 0.71-0.85 0.20-0.23 13.4-16.2 135 6.48-21.6 1.88-2.16 0.961.29-1.56 3.56-4.27 0.23-0.27 25.1-31.4 110 54.0-71.3 0.56-0.751.77-2.01 1.42 0.23 8.95 90 2.16-8.64 0.24-0.32 0.85-1.00 0.17-0.302.68-3.57 80 2.16-3.24 0.40-0.56 3.56-4.27 0.27-0.30 6.26 71 3.24-5.400.43-0.71 0.50-0.77 0.89 64 1.08-3.24 0.57 1.78 59 0.43-0.71 45 40 28 1510* 200 kD-myosin HC; 45 kD-actin; 110 kD-Ca²⁺ ATPase.

The electrophoresis results indicate that only very small amounts ofmembrane protein remain in the supernatant fraction after centrifugationwith the exception of the sample that was treated from pH 7.3 to 10.5 to5 to 10.5 (Sample G).

Example 4 Membrane Sedimentation Behavior in the Presence of pH 3 and pH10.5 “Muscle Supernatants”

The sedimentation behaviors of muscle membranes were investigated in thepresence of “muscle supernatant” (pH=3 or pH=10.5) to determine theeffect of muscle proteins on membrane sedimentation.

pH 3 Procedure

The pH 3 muscle supernatants were prepared as follows:

1. Separated membrane was prepared from 250 g cod muscle tissue, andresuspended in 60 ml histidine buffer.

2. “pH 3 muscle supernatant” was prepared from 120 g cod muscle: Codmuscle was homogenized with 9 times water (citric acid is added to makea final concentration of 1 mM). The homogenate was adjusted to pH 3, andthen centrifuged at 10,000 g for 30 minutes. The resulting supernatantwas termed “pH 3 muscle supernatant.”

3. Volumes of 0 ml (control), 5 ml, 10 ml, 15 ml, and 20 ml of themembrane preparations were each added into five, 120 ml samples of “pH 3muscle supernatant.” Another 10 ml membrane was added into a 120 mlsample of pH 3 HCl solution. After readjusting pH to 3.00±0.05, themixtures are centrifuged at 4,000×g for 15 minutes. The remainingphospholipid (PL) in the resultant supernatants was measured. Theexperiment was performed twice. The percent added membrane remaining inthe supernatant after 4,000 g centrifugation was calculated using thefollowing formula: $\frac{\begin{matrix}{{{PL}\quad{remainaing}\quad{in}\quad{supernatant}\quad{after}\quad{centrifugation}} -} \\{{PL}\quad{from}\quad{``{{muscle}\quad{supernatant}}"}}\end{matrix}}{{PL}\quad{from}\quad{added}\quad{separated}\quad{membrane}}$

The data are summarized in Table 6, below. TABLE 6 MembraneSedimentation Behavior in the Presence of pH 3 “Muscle Supernatants” 10ml No 5 ml 10 ml 15 ml 20 ml membrane membrane membrane membranemembrane membrane added to added added added added added pH = 3 HCl PLfrom120 ml “muscle 38.76 38.76 38.76 38.76 38.76 0 supernatant” or “HCl”(mg) Added membrane 0 37.77 75.54 113.31 151.09 75.54 (mg) PL remainingin 36.72 41.96 58.92 66.04 93.52 1.4 supernatant after centrifugation(mg) Percent added membrane 8.47% 26.69% 24.07% 36.24% <2% remaining inthe supernatant

Typically, when using standard methods, at 10,000×g, 25-40% of themembranes are removed from acidified solubilized protein, i.e., 60-75%of the membranes remain in the supernatant fraction. The percentage ofmembrane that remained in the supernatant fractions in these experimentsvaried with the amount of membrane that was added. In the data presentedin Table 6, 64% to 93% of the added membranes were separated at 4000×gfrom the muscle proteins. This is much more than would be expected to beremoved from the solubilized muscle preparation. Thus, it is not simplythe viscosity and the density differences between the membranes andproteins that are important in the separation step. In theseexperiments, previously separated membrane was added into thesupernatant fraction. Although some of these membranes may havereattached to proteins, it is clear that many more did not and wereeasily separated. The data in Tables 6 (above) and 7 (below) thereforeindicate that part of the reason it is difficult to separate membranesfrom solubilized proteins is that some of the interactions between thecytoskeletal proteins and the membrane fractions are not disrupted bythe process of acidification. The control indicates, as before, that inthe absence of proteins, essentially all of the membrane is sedimentedat pH 3.

pH 10.5 Procedure

The pH 10.5 muscle supernatants were prepared as follows:

1. Separated membrane was prepared from 250 g cod muscle, andresuspended in 60 ml histidine buffer.

2. “pH 10.5 muscle supernatant” was prepared as following: 80 g codmuscle was homogenized with 9 times distilled water for 40 sec (citricacid was added to make a final concentration of 1 mM). The homogenatewas adjusted to pH 10.5. The pH adjusted homogenate was centrifuged at10,000 g for 30 minutes. The resulting supernatant was removed bypipette. The resulting supernatant was termed “pH 10.5 musclesupernatant.”

3. Volumes of 0 ml (control), 5 ml, 10 ml, 15 ml, and 20 ml of themembrane preparations were each added into five, 120 ml samples of “pH 3muscle supernatant.” Another 10 ml membrane was added into a 120 mlsample of pH 10.5 NaOH solution. The resulting mixture was readjusted topH 10.50±0.05. The mixtures were then centrifuged at 4,000 g for 15minutes. The supernatant was removed. Volumes of 15 ml supernatant wereremoved from each sample and used to measure the PL content remaining insupernatant. The experiment was performed twice. Percent added membraneremaining in the supernatant after 4,000 g centrifugation was calculatedas above.

The data are summarized in Table 7, below. TABLE 7 MembraneSedimentation Behavior in the Presence of pH 10.5 “Muscle Supernatants”10 ml membrane No 5 ml 10 ml 15 ml 20 ml added to membrane membranemembrane membrane membrane pH 10.5 added added added added added NaOH PLfrom 120 ml 31.80 31.80 31.80 31.80 31.80 0 “muscle supernatant” or“NaOH” (mg) Added membrane (mg) 0 28.66 57.32 85.78 114.64 57.32 PLremaining in supernatant 32.43 56.00 74.49 90.53 102.87 58.24 aftercentrifugation (mg) % added membrane 83.74% 74.13% 68.23% 61.82% 100%remaining in the supernatant

The results of adding the membrane to solubilized centrifuged proteinextract at pH 10.5 are different from the results obtained at low pH.The percentage of removal ranges roughly from 18-39%. Interestingly, thehigher the amount of membrane that was added, the more of it was removedby the centrifugation process. The data in Table 7 show that thepresence of the solubilized muscle proteins actually improved theremoval of membrane compared to the situation where there was a high pHin the absence of the muscle proteins. There was no removal of membraneby the centrifugation process in the control where no solubilizedprotein was used. This is consistent with the previous data.

Example 5 The Role of Ca²⁺

Calcium ions (Ca²⁺) were used to separate membrane fractions fromproteins at g forces of 4,000 g for 15 minutes. The procedure wasperformed as follows:

Treatment 1: Homogenized muscle+different amount CaCl₂ solution,incubation for 1 hour, adjust pH to 3, centrifugation.

Seven 10 g cod muscle samples were each homogenized with 90 ml distilledwater at speed 5 for 15 seconds. One of the seven samples was used tomeasure the original PL and protein in cod muscle. The other six sampleshad added to them different volumes of 0.5 M CaCl₂ to make the finalCa²⁺ concentration of the mixtures 0 mM (no CaCl₂ added), 0.1 mM (0.02ml CaCl₂ added), 1 mM (0.2 ml CaCl₂ added), 5 mM (1 ml CaCl₂ added), 10mM (2 ml CaCl₂ added), 50 mM (11 ml CaCl₂ added), respectively. Afterstirring, the mixtures were incubated at 0-4° C. in a cold room. After 1hour incubation, the pH of the samples was adjusted to pH 3 beforecentrifugation. After centrifuging at 4,000 g for 15 minutes, the PL andprotein contents remaining in the supernatants were measured.

Treatment 2. Homogenized muscle+different amount CaCl₂ solution,incubation for 1 hour, adjust pH to 10.5, centrifugation.

All the procedures for Treatment 2 were same as in Treatment 1, exceptthe pH of samples was adjusted to 10.5 before centrifugation instead of3.

The original PL content of cod muscle was taken as 100%, and thepercentages of PL remaining in the supernatant after the varioustreatments with different concentrations of CaCl₂ solutions werecalculated. The results are included in Table 8, below. TABLE 8 PercentPL remaining in the Supernatant after Treatment with CaCl₂ No 0.1 mM 1mM 5 mM 10 mM 50 mM Ca²⁺ Ca²⁺ Ca²⁺ Ca²⁺ Ca²⁺ Ca²⁺ Treatment 1 65.47%61.50% 57.30% 46.07% 29.07% 27.30% Treatment 2 74.29% 73.40% 49.37%45.82% 37.98% 10.36%

Next, the original protein content of cod muscle was taken as 100%, andthe percentages of protein remaining in the supernatant after thevarious treatments with different concentrations of CaCl₂ solutions werecalculated. The results are included in Table 9, below. TABLE 9 PercentProtein remaining in the Supernatant after Treatment with CaCl₂ No 0.1mM 1 mM 5 mM 10 mM 50 mM Ca²⁺ Ca²⁺ Ca²⁺ Ca²⁺ Ca²⁺ Ca²⁺ Treatment 191.97% 90.23% 86.83% 86.39% 88.27% 87.09% Treatment 2 92.69% 91.44%90.67% 91.44% 88.29% 49.45%

The data in Tables 8 and 9 indicate that calcium has a positive effecton removal of membranes from the solubilized muscle proteins at bothacid pH (3) or at alkaline pH (10.5). Native concentrations of calciumwhich would be between about 0.1 and 1 mM did not greatly lower theamount of phospholipid at pH 3, but performed moderately well at pH10.5. Higher concentrations, e.g., 10 mM Ca²⁺, removed a considerableamount of the membrane from the solution. Fifty mM performed even betterat the alkaline pH, but it also began to remove some of the muscleproteins.

Example 6 Effect of Incubation Time and Ca²⁺ Concentration on MembraneRemoval

The effect of incubation time with Ca²⁺ before solubilization of themuscle proteins at acid pH was investigated. Two concentrations of Ca²⁺were used, 10 mM and 50 mM which are effective in removing the membrane.

Eight 10 g ground cod samples were weighed and 0.02 g citric acid wasadded to each sample. The samples were homogenized with 90 ml colddistilled water at 5 speed for 25 seconds. One of the eight samples wasused to measure the original PL and protein in cod muscle. The otherseven samples were treated as follows: (1) 0 ml, 2 ml, 11 ml 0.5 M CaCl₂was added to three samples (making the concentration of Ca to be 0 mM,10 mM, 50 mM), the pH of the three samples was adjusted to 3 (0 minutes)as quickly as possible and centrifuged at 4,000 g for 15 minutes. (2) 2ml, 11 ml Ca were added to two samples. The samples were then incubatedfor 30 minutes before adjusted to pH 3 and centrifugation (3) 2 ml, 11ml Ca were added to the other two samples. After 60 minutes ofincubation, the pH of the samples was adjusted to pH 3 and the sampleswere then centrifuged at 4,000 g for 15 minutes. For all treatments, thePL and protein contents remaining in the supernatants were measuredafter centrifugation.

The treatments can be summarized as follows:

Treatment (a): Homogenize muscle (citric acid added beforehomogenization), add CaCl₂ to make the concentration of Ca²⁺ 10 mM and50 mM, incubate for 0 minutes, adjust to pH 3, centrifuge.

Treatment (b): Homogenize muscle (citric acid added beforehomogenization), add CaCl₂ to make the concentration of Ca²⁺ 10 mM and50 mM, incubate for 30 minutes, adjust to pH 3, centrifuge.

Treatment (c): Homogenized muscle (citric acid added beforehomogenization), add CaCl₂ to make the concentration of Ca²⁺ 10 mM and50 mM, incubate for 60 minutes, adjust to pH 3, centrifuge.

The original PL content of cod muscle was taken as 100%, and thepercentages of PL remaining in the supernatant after each treatment wascalculated. The results are included in Table 10, below. TABLE 10Percentages of PL Remaining in the Supernatant No Ca²⁺ 10 mM Ca²⁺ 50 mMCa²⁺ Treatment a 66.50% 43.38% 19.81% Treatment b 26.71% 19.50%Treatment c 19.27% 19.32%

Next, the original protein content of cod muscle was taken as 100%, andthe percentages of protein remaining in the supernatant after eachtreatment was calculated. The results are included in Table 11, below.TABLE 11 Percentages of Protein Remaining in the Supernatant No Ca²⁺ 10mM Ca²⁺ 50 mM Ca²⁺ Treatment a 90.31% 84.12% 69.05% Treatment b 82.72%71.40% Treatment c 81.32% 71.76%

The pH value at the moment just before pH 3 adjustment (the original codhomogenized muscle pH=7.1) was determined. The results are included inTable 12, below. TABLE 12 pH value before adjustment No Ca²⁺ 10 mM Ca²⁺50 mM Ca²⁺ Treatment a 6.52 6.20 5.92 Treatment b 6.22 5.95 Treatment c6.17 5.99

The data above indicates that centrifugation in the absence of calciumremoved less than 40% of the membrane in the solutions of muscleproteins. When 10 mM Ca²⁺ was added, the effectiveness of the removalwas time-dependent, being approximately 57% when there was noincubation, 73% with 30 minutes incubation, and over 80% with 60 minutesincubation. A similar removal of membrane was achieved with 50 mM Ca²⁺to that achieved after 60 minutes with 10 mM Ca²⁺, irrespective of thetime of incubation. It should be noted that a 0 minute incubation doesnot mean 0 minutes of contact between the calcium and the material sinceit takes some time to adjust the pH to a low value. The protein contentremoved at 10 mM Ca²⁺ was greater than that at which there was nocalcium, and was less than what was removed with 50 mM Ca²⁺. Thesetechniques, particularly those at alkaline pH, may serve as afractionation procedure for the protein. High concentrations of calciummay precipitate some of the proteins at both low and high pH values. Thespecific proteins enriched in the precipitates prepared in this way mayprovide unique functional properties. Unlike the case with membraneremoval as determined by phospholipid concentration, the removal ofprotein did not have any significant time dependency. Increasing amountsof Ca²⁺ lowered the pH somewhat.

Example 7 Effect of Calcium in the Absence of Citric Acid at Acid pH

The effect of calcium in the absence of citric acid at acid pH wasinvestigated. Ground cod samples (10 g each sample) were homogenizedwith 90 ml cold distilled water at 5 speed for 25 seconds. One of thesamples was used to measure the original PL and protein in cod muscle.Calcium was added to the samples, making three sets of samples withcalcium concentrations at 0 mM, 0.1 mM, 1 mM, 5 mM, 10 mM, 50 mM in eachset. Three sets were incubated for 0 min, 30 min, and 60 min,respectively, before adjusting the pH to 3. The pH adjusted samples werecentrifuged at 4,000 g for 15 minutes. For all treatments, the PL andprotein contents remaining in the supernatants were measured aftercentrifugation. The treatments are summarized below.

Treatment (a): Homogenize muscle (No citric acid added), add CaCl₂ tomake the concentration of Ca²⁺ 0.1 mM, 1 mM, 5 mM, 10 mM, 50 mM,incubate for 0 minutes, adjust to pH 3, centrifuge.

Treatment (b): Homogenize muscle (No citric acid added), add CaCl₂ tomake the concentration of Ca²⁺ 0.1 mM, 1 mM, 5 mM, 10 mM, 50 mM,incubate for 30 minutes, adjust to pH 3, centrifuge.

Treatment (c): Homogenize muscle (No citric acid added), add CaCl₂ tomake the concentration of Ca²⁺ 0.1 mM, 1 mM, 5 mM, 10 mM, 50 mM,incubate for 60 minutes, adjust to pH 3, centrifuge.

The original PL content of cod muscle was taken as 100%, and thepercentages of PL remaining in the supernatant after each treatment wascalculated. The results are included in Tables 13, below. TABLE 13Percentages of PL Remaining in the Supernatant 0 mM 0.1 mM 1 mM 5 mM 10mM 50 mM a 89.60% 86.03% 85.64% 80.63% 64.54% 51.45% b 91.83% 89.47%85.24% 78.53% 64.05% 55.67% c 90.88% 85.83% 85.16% 77.96% 65.50% 54.20%

Next, the original protein content of cod muscle was taken as 100%, andthe percentages of protein remaining in the supernatant after eachtreatment was calculated. The results are included in Table 14, below.TABLE 14 Percentages of Protein Remaining in the Supernatant 0 mM 0.1 mM1 mM 5 mM 10 mM 50 mM a 95.35% 94.78% 93.23% 94.39% 94.42% 86.89% b95.23% 94.37% 93.43% 92.00% 92.16% 87.73% c 97.61% 97.42% 92.40% 91.50%92.91% 88.32%

The pH value at the moment just before pH 3 adjustment (the original codhomogenized muscle pH=7.1) was determined. The results are included inTable 15, below. TABLE 15 pH Value before Adjustment 0 mM 0.1 mM 1 mM 5mM 10 mM 50 mM pH 7.19 7.08 6.95 6.74 6.60 6.50

The data above indicate that treatments with CaCl₂ gave some improvementwith increasing Ca²⁺ concentration, which was not time-dependent.Membrane removal is improved even further when the process is carriedout in the presence of citric acid.

Example 8 The Effect of Citric Acid

Whether the membrane sedimentation effect was due to citric acid orsimply H⁺ provided by the citric acid was investigated. Nine 10 g groundcod samples were weighed. 0.02 g citric acid was added to four of thenine samples. The samples were homogenized with 90 ml cold distilledwater at 5 speed for 25 seconds. One of the nine samples (no citric acidadded) was used to measure the original PL and protein in cod muscle.The other eight samples were treated as follows: (1) For the foursamples without adding citric acid, HCl was added to adjust their pH to6.50. One of them was adjusted to pH 3 immediately to act as a control.Two ml of 0.5 M CaCl₂ was added to the other three samples (making theconcentration of Ca²⁺ 10 mM), and then incubated for 0, 30, and 60minutes before adjusting the pH to 3.

(2) For the four samples with citric acid added (pH around 6.50), one ofthem was adjusted to pH 3 immediately to act as control. Two ml of 0.5 MCaCl₂ was added to the other three samples (making the concentration ofCa²⁺ to be 10 mM), and then incubated for 0 minutes, 30 minutes, 60minutes before adjusting to pH 3. All the resultant samples werecentrifuged at 4,000 g for 15 minutes. For all the treatments, the PLand protein contents remaining in the supernatants were measured aftercentrifugation. The treatments used in the investigation are summarizedbelow.

Treatment (a) (with citric acid): Homogenize muscle (20 mg citric acidadded before homogenization, which made the pH of homogenized muscle tobe around 6.50), add CaCl₂ solution to make the concentration of Ca²⁺ 10mM, incubate for 0 minutes, 30 minutes, 60 minutes, adjust to pH 3 byHCl, centrifuge.

Treatment (b) (without citric acid): Homogenize muscle, adjust the pH ofhomogenized muscle to 6.50 with HCl, add CaCl₂ solution to make theconcentration of Ca²⁺ 10 mM, incubate for 0 minutes, 30 minutes, 60minutes, centrifuge.

The original PL content of cod muscle was taken as 100%, and thepercentages of PL remaining in the supernatant after each treatment wascalculated. The results are included in Tables 16, below. TABLE 16Percentages of PL Remaining in the Supernatant Incubation No ions timecontrol 0 min 30 min 60 min Treatment A 69.65% 60.12% 57.96% 60.92%Treatment B 68.00% 31.67% 28.28% 25.55%

Next, the original protein content of cod muscle was taken as 100%, andthe percentages of protein remaining in the supernatant after eachtreatment was calculated. The results are included in Table 17, below.TABLE 17 Percentages of Protein Remaining in the Supernatant IncubationNo Ca time control 0 min 30 min 60 min Treatment A 94.90% 93.68% 90.82%88.06% Treatment B 92.69% 84.96% 85.77% 83.30%

With regard to pH, the pH of the homogenized muscle was 7.10. Beforeadding Ca²⁺ (lowered by citric acid or HCl) the pH was 6.50. Afteradding Ca²⁺ (2 ml 0.5M Ca²⁺), the pH was 6.20. The data indicates thatefficient removal of membrane from the solubilized muscle solutiondepends not just on the H⁺ concentration (pH), but also on the acidutilized.

Example 9 The Role of Mg⁺²

The membrane sedimentation effect of MgCl₂ was investigated. Five 10 gground cod samples were weighed. 0.02 g citric acid was added to four ofthe five samples. The samples were homogenized with 90 ml cold distilledwater at 5 speed for 25 seconds. One of the five samples (no citric acidadded) was used to measure the original PL and protein in cod muscle.The other four samples were treated as follows: (1) one sample(control): adjusted to pH 3 by HCl; (2) three samples: added 2 ml 0.5MMgCl₂ to make the concentration of Mg 10 mM, incubated for 0 minutes, 30minutes, 60 minutes respectively. After incubation, all samples wereadjusted to pH 3. All samples were centrifuged at 4,000 g for 15minutes. For all the treatments, the PL and protein contents remainingin the supernatants were measured after centrifugation. The treatmentused in the investigation is summarized below.

Treatment: Homogenized muscle (20 mg citric acid added beforehomogenization, which made the pH of homogenized muscle to be around6.50-6.60), add MgCl₂ solution to make the concentration of Mg 10 mM,incubate for 0 minutes, 30 minutes, 60 minutes, adjust to pH 3 by HCl,centrifuge.

The original PL content of cod muscle was taken as 100%, and thepercentages of PL remaining in the supernatant after each treatment wascalculated. The results are included in Tables 18, below. TABLE 18Percentages of PL Remaining in the Supernatant Incubation time No Mgcontrol 0 min 30 min 60 min Mg Treatment 66.50% 52.73% 35.56% 34.09%

Next, the original protein content of cod muscle was taken as 100%, andthe percentages of protein remaining in the supernatant after eachtreatment was calculated. The results are included in Table 19, below.TABLE 19 Percentages of Protein Remaining in the Supernatant Incubationtime No Mg control 0 min 30 min 60 min Mg Treatment 90.72% 91.03% 88.78%87.63%

These data indicate that magnesium ion has a time-dependent effect onremoval of membrane from the solubilized muscle proteins at pH 3.Time-dependency is clearer than it was in the case of calcium, whereasthe percent removal (about 65% after 30 and 60 minutes) was somewhatless than is typically removed in the presence of CaCl₂.

Example 10 Effect of Ca²⁺ and Mg²⁺ Added after Solubilization ofProteins at pH 3

The effect of adding calcium and magnesium chlorides after adjusting thesolubilized proteins to pH 3 in the presence of citric acid, rather thanbefore solubilization, was investigated. Eight 10 g ground cod sampleswere weighed. 0.02 g citric acid was added to seven of the eightsamples. The samples were homogenized with 90 ml cold distilled water at5 speed for 25 seconds One of the eight samples (no citric acid added)was used to measure the original PL and protein in cod muscle. The otherseven samples were treated as follows: (1) one sample (Control):adjusted to pH 3 with HCl, then centrifuged; (2) three samples: adjustedpH 3 with HCl, then added 2 ml 0.5 M MgCl₂ to make the concentration ofMg²⁺ 10 mM, incubated for 0 minutes, 30 minutes, 60 minutes beforecentrifuging; (3) three samples: adjusted to pH 3 with HCl, then added 2ml 0.5 M CaCl₂ to make the concentration of Ca²⁺ 10 mM, incubated for 0minutes, 30 minutes, 60 minutes, respectively before centrifuging. Allsamples were centrifuged at 4,000 g for 15 min. For all the treatments,the PL and protein contents remaining in the supernatants were measuredafter centrifugation. The treatments used in the investigation aresummarized below.

Treatment (a): Homogenize muscle (20 mg citric acid added beforehomogenization, which made the pH of homogenized muscle to be around6.50), adjust to pH 3 with HCl, add MgCl₂ solution to make theconcentration of Mg 10 mM, incubate for 0 minutes, 30 minutes, 60minutes, centrifuge.

Treatment (b): Homogenize muscle (20 mg citric acid added beforehomogenization, which made the pH of homogenized muscle to be around6.50), adjust to pH 3 with HCl, add CaCl₂ solution to make theconcentration of Ca 10 mM, incubate for 0 minutes, 30 minutes, 60minutes, centrifuge.

The original PL content of cod muscle was taken as 100%, and thepercentages of PL remaining in the supernatant after each treatment wascalculated. The results are included in Table 20, below. TABLE 20Percentages of PL Remaining in the Supernatant Incubation No ions timecontrol 0 min 30 min 60 min Treatment A 74.50% 74.10% 70.36% 72.25%Treatment B 74.50% 69.38% 72.31% 71.21%

Next, the original protein content of cod muscle was taken as 100%, andthe percentages of protein remaining in the supernatant after eachtreatment was calculated. The results are included in Table 21, below.TABLE 21 Percentages of Protein Remaining in the Supernatant IncubationNo Ca time control 0 min 30 min 60 min Treatment A 92.35% 91.92% 90.93%88.66% Treatment B 92.35% 88.74% 89.15% 90.90%

The data above indicate that the addition of CaCl₂ or MgCl₂ solutions tothe solubilized muscle proteins at acidic pH did not remove the membranefraction.

Example 11 Aggregated Cellular Membranes Exhibit Reduced Susceptibilityto Hemoglobin-Catalyzed Oxidation

Whether aggregation of cellular membranes renders them resistant tohemoglobin-catalyzed oxidation was investigated. The results of theexperiments are provided in Tables 22 and 23, below.

Fish Supply

Cod fish (Gadus morhua) was obtained from day boats in Gloucester, Mass.The fish was transported in ice to the laboratory and was filleted,skinned, and minced. All dark muscle was removed.

Isolated Membrane Model System

The method of McDonald and Hultin (J. Food Sci. 52, 15-21 (1987)) wasmodified for isolating membranes. Four volumes of a cold 0.1 M HEPESbuffer (pH 7.5) were added to the minced muscle and homogenized using aPolytron® PT 10-35 (Brinkman Instruments, Westbury, N.Y.) for 30 secondsat speed 5. The pH of the homogenate was adjusted to 7.5 and centrifugedat 10,000×g for 20 minutes at 5-10° C. in a Beckman L8-55MUltracentrifuge (Beckman Instruments, Palo Alto, Calif.). Thesupernatant was recentrifuged at 130,000×g for 30 minutes at 5-10° C.The resulting sediment was suspended in a cold 0.12 M KCL and 5 mMhistidine (pH 6.8) buffer with a Potter homogenizer and stored at −80°C. for no longer than one week.

The model system was formed with a final concentration of 0.7 mg ofmembrane protein/ml in 0.12 KCL and 5 mM histidine to the pH valuesindicated. Incubations were carried out in a shaking water bath at 6° C.in air in 25 ml Erlenmeyer flasks. Reactions were initiated by theaddition of hemoglobin. The pH was adjusted before adding hemoglobin andit was checked after adding hemoglobin.

Membranes of Washed Cod Model System

The washed cod muscle was prepared by an adaptation of the procedure ofRichards and Hultin (2000). A 50 mM sodium phosphate, 0.12 M KCL and 5mM histidine solution were used in the washed cod to modify ionicstrength and modify pH. The final washed cod was frozen at −80° C. Themuscle was thawed in a sealed plastic bag under running cold water.After the pH was adjusted, the moisture was measured. Streptomycinsulfate (200 ppm) was added to inhibit microbial growth. The finalmoisture was adjusted to 88%. A volume of hemoglobin stock solution wasadded to a final concentrate of 3 μmol per kg of washed cod muscle. Inthe control the hemoglobin solution was replaced by distilled water. Thesamples were incubated at 2° C.

Preparation of Cod Hemolysate

The cod blood was collected from cod frames obtained after filleting inthe rigor state. The blood was taken from the caudal vein after the tailof the fish frame was cut off. Fish blood was drawn from the openedcaudal vein by using a transfer glass pipette rinsed with 150 mM NaClwith sodium heparin solution (30 units/ml). The blood was immediatelymixed with approximately 1 volume of saline sodium heparin solution.Hemolysate was prepared following the procedure of Fyhn et al. (Comp.Biochem. Physiol. 62A, 39-66 (1979)) modified by Richards and Hultin (J.Agric. Food Chem. 4, 314103147 (2000)). Four volumes of ice-cold 290 mMNaCl in 1 mM Tris, pH 8.0, were added to heparinized blood.Centrifugation was done at 700×g for 10 min at 4° C. using a table topclinical centrifuge (IEC, Needham Heights, Mass.). Plasma was thenremoved. Red cells were washed by suspending three times in 10 volumesof the above buffer and centrifuging at 700×g. Cells were lysed in 3volumes of 1 mM Tris, pH 8.0 for 1 h. One-tenth volume of 1 M NaCl wasthen added to aid in stromal removal before centrifugation at 28,000×gfor 15 min at 4° C. in a Beckman Ultracentrifuge Model L5-65B (BeckmanInstruments Inc., Palo Alto, Calif.). Hemolysate was stored at −80° C.

Hemoglobin (Hb) levels were quantified with modifications of the methodof Brown (J. Biol. Chem. 236, 2238-2240 (1961)). Approximately 1 mg ofsodium dithionite was added to the extract and mixed in a cuvette. Thenthe sample was bubbled with carbon monoxide gas (Matheson Gas,Gloucester, Mass.) for 30 sec. The sample was then scanned from 440 to400 nm (Soret) against a blank that contained only buffer using a ModelU-3110 double-beam spectrophotometer (Hitachi Instruments, Inc., SanJose, Calif.). The peak was recorded. Standard curves were constructedusing bovine hemoglobin standard in 50 mM Tris, pH 8.6 buffer on 50 mMTris, 200 mM NaCl, pH 6.95 buffer. In a separate procedure, when verylow levels of hemoglobin were present, a portion of the extract wasmixed with approximately 1 mg sodium dithionite in a cuvet. The samplewas immediately scanned spectrophotometrically against a reference withno sodium dithionite. The difference in absorbance of the peak (432 nm)and valley (410 nm) of the spectrum was used to measure hemoglobin.

Thiobarbituric Acid Reactive Substances (TBARS) Analyses

Thiobarbituric acid reactive substances (TBARS) were determined using amuscle extraction procedure in which EDTA (0.1%) and propyl gallate(0.1%) were added to the extraction solution (7.5% trichloroacetic acid,TCA) to lessen development of TBARS during the analytical procedure. Amodification included using 1 g of sample and extracting with 6 ml ofTCA solution by homogenization with a Tissue Tearor™ model 986-370 athigh speed (Biospec Products Inc., Bartlesville, Okla.). For thestandard curve an extinction coefficient of 1.30×10⁵ M⁻¹cm⁻¹ wasdetermined using tetraethoxypropane standard. The TBARS data areexpressed as μmol malonaldehyde per kg tissue or per kg lipid.

Determination of Protein Content

Protein content was determined by the method of Markwell et al. (Anal.Biochem. 87, 206-210 (1978)). Bovine albumin was used.

Table 24, below, demonstrates that hemoglobin-catalyzed oxidation ofisolated cod sarcoplasmic reticulum lipids is slowest at pH 3.5. This isin contrast to what was observed in washed cod muscle where theoxidation is rapid at acid pH values up to 6.8 (see Table 23, below).The last column in Table 22 shows that even after re-adjusting the pH to8 from 3.5, a large proportion of the antioxidation effectiveness isretained. As indicated in other examples described above, acid pHisolated membrane is sedimented easily at low centrifugal force,indicating that the membranes are aggregated. TABLE 22 Effect of pH onHemoglobin-Catalyzed Oxidation of Isolated Cod Sarcoplasmic_ReticulumLipids pH, TBARS as nmol MDA/mg membrane proteins time (h) 3.5 4.5 6.06.8 8.0 3.5¹ increase to 8.0 0 0 0 0 0 0 0 1.5 0 2.7 1.3 10.2 31 1.8 2.50.8 20.6 22.6 78.3 72 6.4 3.0 0 18.8 66.0 87.6 80.1 8.1 3.5 7.5 36.488.8 95.0 83.3 12.6 4.0 15.6 49.8 97.5 97.6 84.0 26.6 5.0 27.8 64.9108.5 101.7 85.2 53.4 6.0 57.8 87.8 112.8 99.4 85.5 51.7¹Membranes were stored for 30 minutes at pH 3.5 before being brought topH 8.0. 6 μM hemoglobin was added at a temperature of 2° C.MDA = malonaldehyde.

TABLE 23 Effect of pH on Hemoglobin-Catalyzed Oxidation of the CodMembrane Lipids of Washed Cod Muscle pH, TBARS as μmol MDA/kg washed codtime, h 3.5 6.8 7.6 3.5¹ increase to 8.0 0 5.7 0 0 4.9 6 185.0 193.8 5.318.2 12 267.1 273.6 62.3 59.6 16 273.2 285.5 141.0 81.5 28 262.0 269.5261.8 155.9 43 283.3 300.7 257.8 167.7¹Washed cod was stored at pH 3.5 for 1.5 h before adjusting to pH 7.6. 3μM hemoglobin was used. Other conditions as in Table 22.

Table 23 demonstrates that the non-isolated cellular membranes of washedcod muscle oxidize rapidly at acid pH values up to 6.8.

The model systems used in the present example are recognized in the artas involving “accelerated” oxidation conditions. The oxidationconditions are “accelerated” because the systems are free of naturalantioxidants and include a high concentration of added pro-oxidant(hemoglobin). Accordingly, membrane oxidation in the accelerated systemwill occur more rapidly than it would in a non-accelerated (i.e.,natural) system. Skilled practitioners will appreciate, however, thatthe relative rates of oxidation in accelerated and non-acceleratedsystems are similar. For example, a four-fold decrease in the rate ofoxidation in the accelerated system would correspond to an approximatelyfour-fold decrease in the rate of oxidation in a non-accelerated (i.e.,natural) system.

Skilled practitioners will recognize that the data presented abovesuggest that aggregated cellular membranes are substantially less proneto oxidation than non-aggregated membranes, and that proteincompositions possessing such oxidation-resistant membranes may have anextended shelf-life as compared to protein compositions containingreadily-oxidizable membranes.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of preparing an edible protein composition with reducedoxidation protential from animal muscle, the method comprising: (a)obtaining a mixture comprising minced or ground animal muscle; (b)adding an amount of a polyvalent, food-grade cation to the mixturesufficient to separate cellular membranes from cytoskeletal proteins inthe animal muscle; and (c) treating the mixture to reduce the oxidationpotential of the separated cellular membranes.
 2. The method of claim 1,wherein the polyvalent, food-grade cations are calcium or magnesiumions, and wherein the concentration of calcium or magnesium ions in themixture is in a range of about 0.1 mM to about 50 mM after the addition.3. The method of claim 1, wherein step (c) comprises aggregating atleast a portion of the separated cellular membranes in the mixture toreduce the total separated membrane surface area, thereby reducing theoxidation potential.
 4. The method of claim 3, further comprising (d)dewatering the mixture.
 5. The method of claim 1, wherein step (c)comprises removing the separated cell membranes from the mixture,thereby reducing the oxidation potential.
 6. The method of claim 1,wherein step (c) comprises incubating the mixture to allow the separatedmembranes to aggregate, thereby reducing the oxidation potential, andwherein the method further comprises (d) adjusting the pH of the mixtureto solubilize at least a portion of the protein in the mixture.
 7. Themethod of claim 6, further comprising (e) removing the aggregatedseparated cell membranes from the solubilized protein.
 8. The method ofclaim 7, further comprising (f) collecting the solubilized protein. 9.The method of claim 3, wherein step (c) comprises adding an acid to themixture.
 10. The method of claim 6, wherein step (d) comprises adding anacid to the mixture.
 11. The method of claim 9, wherein sufficient acidis added to the mixture to lower the pH to below about 3.5.
 12. Themethod of claim 6, wherein step (d) comprises adding a base to themixture.
 13. The method of claim 12, wherein sufficient acid is added tothe mixture to raise the pH to greater than about 10.0.
 14. The methodof claim 1, wherein the polyvalent food-grade cation is calciumchloride.
 15. The method of claim 1, wherein the polyvalent food-gradecation is magnesium chloride.
 16. The method of claim 1, wherein step(c) comprises incubating the mixture for up to 60 minutes following step(b).
 17. The method of claim 1, further comprising adding an organicacid to the mixture before, during, or after step (b).
 18. The method ofclaim 17, wherein the organic acid is selected from the group consistingof citric, malic, maleic, fumaric, and tartaric acid.
 19. The method ofclaim 7, wherein step (e) is performed by centrifugation.
 20. The methodof claim 19, wherein the centrifugation is performed at from about 500×gto about 10,000×g.
 21. The method of claim 7, wherein step (e) isperformed by precipitation.
 22. The method of claim 3, whereinaggregating is performed by adding to the mixture an aggregant selectedfrom the group consisting of carrageenan, algin, demethylated pectin,gum arabic, chitosan, polyethyleneimine, spermine, spermidine, calciumsalt, magnesium salt, sulfate, phosphate, and polyamine.
 23. The methodof claim 6, wherein step (c) further comprises adding to the mixture anaggregant selected from the group consisting of carrageenan, algin,demethylated pectin, gum arabic, chitosan, polyethyleneimine, spermine,spermidine, calcium salt, magnesium salt, sulfate, phosphate, andpolyamine.
 24. The method of claim 4, wherein dewatering is performed bycentrifuging the mixture.
 25. The method of claim 4, wherein dewateringis performed by filtering the mixture.
 26. The method of claim 4,wherein dewatering is performed by pressing the mixture.
 27. The methodof claim 1, further comprising homogenizing the animal muscle betweensteps (a) and (b).
 28. The method of claim 1, wherein step (b) and (c)are performed simultaneously by adding a sufficient amount of calcium ormagnesium ions to cause both separation of cellular membranes andcytoskeletal proteins and aggregation of the membranes.
 29. An edibleprotein composition with reduced oxidation potential comprising mincedor ground animal muscle, wherein the composition comprises a cellularmembrane content of less than 40% of a cellular membrane content in asample of the animal muscle as removed from the animal prior to mincingor grinding.
 30. The edible protein composition of claim 29, wherein theprotein composition has a cellular membrane content less than 25% ofthat in the animal muscle.
 31. The edible protein composition of claim29, wherein the protein composition has a cellular membrane content lessthan 20% of that in the animal muscle.
 32. The edible proteincomposition of claim 29, wherein the protein composition has a cellularmembrane content less than 10% of that in the animal muscle.
 33. Theedible protein composition of claim 29, wherein the protein compositionhas a cellular membrane content less than 5% of that in the animalmuscle.
 34. An edible protein composition with reduced oxidationpotential comprising minced or ground animal muscle, wherein thecomposition comprises a cellular membrane content approximately equal tothe cellular membrane content in a sample of the animal muscle asremoved from the animal prior to mincing or grinding, wherein at least60% of the cellular membranes in the edible protein composition areseparated from cytoskeletal proteins and are in aggregated form, asdetermined using microscopy.
 35. An edible protein composition withreduced oxidation potential produced by the process of claim 1.